1. Field of the Invention
This invention relates to tissue engineering, and more particularly to a system and method for producing engineered bone, ligament, and bone-ligament constructs.
2. Background Art
Bone is a vascularized tissue composed of a number of different types of cells. The tissue is predominately made up of a mineralized type I collagen matrix, and crystals within the mineralized matrix are composed of hydroxyapatite, a form of calcium phosphate. The three different types of cells in bone are osteoblasts, osteocytes, and osteoclasts. These cells each have different functions that allow bone tissue to continually remodel itself. Osteoblasts are the cells involved in the deposition and mineralization of type I collagen. These cells are round in morphology with cytoplasmic projections. Once the osteoblast is fully surrounded by mineralized matrix, it differentiates into an osteocyte. Osteocytes are cells that sit in open lacunae within mineralized bone. The functions of an osteocyte are to both resorb bone and to deposit new bone. Osteocytes are connected to other cells via cytoplasmic projections that can travel through channels within the mineralized matrix. Finally, osteoclasts are large multinucleated cells with large vacuoles that are involved in the bone resorption. Osteoclasts have two different types of plasma membranes: clear zones and ruffled borders. The ability of bone to remodel itself allows it to change its architecture and constitution (e.g. local density) with changes in its loading environment. Also, when fractured or inflicted with a small defect, bone can easily heal by the combination of processes of collagen deposition, mineralization, and resorption.
Ligaments are dense, relatively avascular connective tissues of the musculoskeletal system that help control joint motion, along with muscle. These tissues connect one bone to another and function to provide mechanical stability in joints, serve as a guide to joint motion, and prevent excess motion. About 80% by volume of ligament tissue is composed of longitudinally aligned collagen bundles. Most of the collagen is type I, however, type III is also present, as is elastin. Fibroblasts are the cellular component in ligaments and make up approximately 20% of the adult tissue volume. These cells attach to the individual collagen bundles and are elongated longitudinally.
The interface between bone and ligament is referred to as an enthesis. The purpose of the enthesis tissue is to transmit loads with high fidelity over a minimal volume of tissue from the compliant ligament to the stiff bone at the bone-ligament interface. This tissue is composed of four different zones that aid in the transition between the two vastly different tissues. The four zones of the enthesis are ligament, unmineralized fibrocartilage, mineralized fibrocartilage, and bone. The transition from ligament to unmineralized fibrocartilage is gradual, whereas a distinct boundary exists between unmineralized and mineralized fibrocartilage in adult tissue. This boundary is termed a tidemark and can be identified using hematoxylin and eosin (H and E) staining due to its extreme basophilic nature (Claudepierre and Voisin, Joint Bone Spine 72: 32, 2005; Benjamin et al., J Anat 208: 471, 2006). Fibrocartilage zones are composed of type II collagen and proteoglycans such as aggrecan, biglycan and decorin. The cells in fibrocartilage have the phenotype of chondrocytes, round and arranged in pairs or rows within lacunae. There are no molecular markers that are unique to this type of tissue, however, fibrocartilage, and in general the enthesis, is generally characterized by the presence of type II collagen due to the fact that this protein is not present in the neighboring ligament and bone tissues (Waggett et al., Matrix Biol 16: 457, 1998).
There are approximately one million surgeries each year in the United States that require bone and ligament grafts to replace tissue damaged by disease or extensive trauma. Several limitations are associated with grafting, such as graft availability, donor site morbidity, and immune rejection. Because of these complications, strategies are being developed to engineer bone and ligament tissue in vitro.
Current approaches to engineer bone and ligament involve the design of a three-dimensional (3D) scaffold that promote the differentiation and proliferation of osteogenic or fibroblastic cells and the deposition and mineralization of an osteogenic or fibroblastic extracellular matrix (ECM). The scaffold design rubrics also include the ability to withstand physiological loads in vivo and either the eventual incorporation into the native tissue or degradation during the course of tissue development (Salgado et al., Macromol Biosci 4: 743, 2004). Polymers such as poly(lactic-co-glycolic acid), poly(propylene fumarates), and poly(caprolactones) provide a matrix that promotes cell adhesion and migration, allow for the deposition and mineralization of ostcogcnic ECM in vitro, and have predictable degradation rates, but lack the mechanical properties needed to withstand the loads placed on natural bone in vivo (Ishaug et al, J Biomed Mater Res 36: 17, 1997; Vehof et al., J Biomed Mater Res 60: 241, 2002; Peter et al., J Biomed Mater Res 43: 422, 1998).
Hydroxyapatite and b-tricalcium phosphates are ceramics used for bone scaffolding that also promote cell adhesion and proliferation and, when implanted, have shown positive results in regards to bone regeneration in vivo. However, the brittle nature of ceramics inhibits their use in healing large defects (Salgado, 2004; Ducheyne and Qiu, Biomaterials 20: 2287, 1999). Polymer-ceramic composite scaffolds such as calcium phosphate salts embedded in poly(caprolactones) have been designed to mitigate the problems with using each material alone, but a significant percentage of cells fails to attach to the composite scaffold due to limited surface-to-volume ratio (Zhou et al., Polym Int 56: 333, 2007; Zhou et al., Biomaterials 28: 814, 2007). Single layer cell sheets grown from bone marrow stromal cells (BMSC) and wrapped around composite scaffolds have recently been shown to form constructs that resemble bone in vitro and in vivo (Zhou et al., Biomaterials 2007). However, this method still involves the use of an exogenous scaffolding that must incorporate into native tissue. Therefore, while scaffolding strategies appear to promote osteogenic or fibroblastic cell growth, limitations such as immune rejection, degradation, and nonphysiological mechanical properties of the scaffold need to be considered when used for bone and ligament repair.